Biflavone-zinc complex, preparation method and application thereof

ABSTRACT

The present invention discloses a biflavone-zinc complex, a preparation method and application thereof. In the present invention, using the amentoflavone as the ligand and the zinc ion as the central ion, the amentoflavone-zinc complex is obtained by reaction, and the structure of the complex is characterized by infrared spectroscopy, UV-vis absorption spectroscopy and high resolution mass spectrometry. Meanwhile, the antitumor and antioxidant activities of the amentoflavone-zinc complex are studied in the present invention. MTT method shows that the amentoflavone-zinc complex has better antitumor activity, and the antitumor activity thereof is stronger than that of the amentoflavone. Pyrogallol auto-oxidation method and ABTS method show that the antioxidative activity of the amentoflavone-zinc complex is stronger than that of the amentoflavone itself. The formation of the flavonoid-zinc complex enhances the antitumor and anti-oxidation activities of the amentoflavone, and is expected to be used in the development of drugs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase entry of InternationalApplication No. PCT/CN2018/076739, filed on Feb. 13, 2018, which isbased upon and claims priority to Chinese Patent Application No.201710097624.3, filed on Feb. 22, 2017, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention belongs to the fields of organic synthesis andmedical technology, specifically relating to a biflavone-zinc complex, apreparation method and an application thereof.

BACKGROUND

Biflavonoid compounds are unique chemical components of gymnosperms suchas ginkgo, Selaginella tamariscina, etc., which have biologicalactivities such as anti-oxidation, anti-inflammatory, anti-viral,antitumor and the like. Among them, amentoflavone (Ame) is a more commonone of the biflavonoid compounds with a structural formula shown asfollows.

Sun et al. has researched and found that the amentoflavone can increasethe expression of anti-cancer genes by activating hPPARγ, therebyachieving the effect of inhibiting breast cancer cells and cervicalcancer cells (Lee E., Shin S., Lee J. et al. Cytotoxic activities ofamentoflavone against human breast and cervical cancers are mediated byincreasing of PTEN expression levels due to peroxisomeproliferator-activated receptor γ activation [J]. Bulletin of the KoreanChemical Society, 2012, 33(7): 2219-2223.). Chen et al. has researchedand found that the amentoflavone blocks the formation of blood vesselsand the metabolism of cancer cells through inhibiting the activity offactor NF-kappa B, achieving the purpose of inhibiting the growth oftumor cells (Chen J. H., Chen W. L., Liu Y C. A amentoflavone inducesanti-angiogenic and anti-metastatic effects through suppression ofNF-kappa B activation in MCF-7 cells [J]. Anticancer Research, 2015,35(12):6685-6693.). Lee et al. has researched and found that theamentoflavone may inhibit UVB-induced matrix metalloproteinaseexpression, thereby exerting anti-oxidation and anti-radiation effects(Lee C. W., Na Y, Park N., et al. Amentoflavone inhibits UVB-inducedmatrix metalloproteinase-1 expression through the modulation of AP-1components in normal human fibroblasts [J]. Applied Biochemistry andBiotechnology, 2012, 166:1137-1147.). Zhang et al. has researched andfound that the amentoflavone and ginkgetin have certain antioxidantactivity and strong ability to scavenge DPPH free radicals (Zhang Y. P.,Shi S. Y, Wang Y. X., et al. Target-guided isolation and purification ofantioxidants from Selaginella sinensis by offline coupling of DPPH-HPLCand HSCCC experiments [J]. Journal of Chromatography B, 2011,879:191-196.). Li et al. has researched and showed that theamentoflavone has an antioxidant activity, which may effectivelyscavenge free radicals such as OH⁻., O²⁻., DPPH., ABTS⁺., etc., andprotect DNA from oxidative damage caused by OH⁻. (Li X. C., Wang L., HanW. J., et al. Amentoflavone protects against hydroxyl radical-inducedDNA damage via antioxidant mechanism [J]. Turkish Journal ofBiochemistry-Turk Biyokimya Dergisi, 2014, 39(1):30-36.).

The coordination chemistry of traditional Chinese medicines shows thatcomplex equilibria exists in the complexes formed by the reactionbetween trace elements and organic compounds, so the biologicalactivities of the original components can be exhibited. Moreover, sincethe synergy and antagonism that exists in trace elements, organiccomponents, complexes and a combination thereof may weaken or enhancethe biological activities of the original components, new biologicalactivities may also be generated (Cao Zhiquan. New thoughts of studyingmaterial basis and mechanism of the efficacy of traditional Chinesemedicine (1)-Study on the relationship between the speciation andbiological activity of chemical species in traditional Chinese medicine[J]. Journal of Shanghai University of Traditional Chinese Medicine,2000, 14(1):36-39.). For example, Zhou et al. has researched and foundthat the quercetin rare earth complex has higher ability to scavenge O₂⁻ than the quercetin. The quercetin rare earth complex can inhibit avariety of tumors, and antitumor activity thereof is higher than that ofthe quercetin. Wherein, the complex has a higher inhibitory effect onbladder tumor cells, while the quercetin does not have such effect (ZhouJ., Wang L. F., Wang J. Y, et al. Synthesis, characterization,antioxidative and antitumor activities of solid quercetin rareearth(III) complexes [J]. Journal of Inorganic Biochemistry, 2001,83:41-48.).

However, to date, studies on the synthesis of the biflavone complex andthe biological activity thereof have not been reported.

SUMMARY

Objectives of the Present Invention: in view of the deficiencies in theprior art, one objective of the present invention is to provide anamentoflavone-zinc complex, which meets the application needs ofantitumor and anti-oxidation drugs. Another objective of the presentinvention is to provide a preparation method of the above-mentionedbiflavone-zinc complex. A further objective of the present invention isto provide the application of the biflavone-zinc complex.

Technical Solution: in order to achieve the above objectives, thetechnical solution of the present invention is as follows.

The biflavone-zinc complex has the following structural formula:

X is NO₃ ⁻ or Cl⁻.

A preparation method of a biflavone-zinc complex is as follows: a zincsalt is dissolved in an alcohol and then added into a biflavonedissolved in an alcohol, the pH is controlled to 5-7, under heating andstirring, the reaction is continued for 2-5 h to form a precipitate; theprecipitate is filtered, and then washed with alcohol and water, afterthat, recrystallized using dimethyl sulfoxide as a solvent, and finallydrying is performed to obtain the biflavone-zinc complex.

The biflavone is an amentoflavone, but is not limited to theamentoflavone, and generally refers to biflavonoid compounds having 5-OHand 4-C═O, or having 5″-OH and 4″-C═O.

The zinc salt is an alcohol-soluble zinc salt such as zinc nitrate, zincchloride, etc.

The solvent is ethanol, methanol and methanol/ethanol aqueous solutionof various concentrations, etc.

The pH is adjusted with an alkali alcohol solution, and the alkali usedin the alkali alcohol solution includes common bases such as sodiumhydroxide, potassium hydroxide, aqueous ammonia, sodium ethoxide, sodiummethoxide, etc.

During the reaction, the heating temperature is 30° C.-50° C., and thereaction time is 2-5 h.

The molar ratio of the biflavone to zinc ion in the solution is 2-2.5:1.

The solvent used in the recrystallizing is dimethyl sulfoxide, and thedrying method is freeze-drying, low temperature vacuum-drying, etc.

The biflavone-zinc complex is used in the preparation of an antitumordrug and/or an antioxidant drug.

Beneficial Effects: compared with the prior art, the amentoflavone-zinccomplex is first synthesized by the present invention. The antitumoractivity of the Ame-Zn complex is studied by the MTT method, and theresults show that the ability of the Ame-Zn complex to inhibit hepatomacells (HepG2) and cervical cancer cells (HeLa) is stronger than that ofthe Ame itself. Through UV-vis absorption spectroscopy, fluorescencespectroscopy and viscosity methods, it shows that the mechanism ofantitumor activity of the Ame-Zn complex may be that the complex isinserted into DNA in an intercalation manner, causing apoptosis. Throughthe pyrogallol auto-oxidation method and ABTS method, it shows that theAme-Zn complex has stronger ability to scavenge free radicals than theAme itself, indicating that the antioxidant activity of the complex isstronger than that of Ame, which is conducive to the further developmentof the biflavonoid compounds, provides a basis for the research of newdrugs and contributes to the development of human health.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an IR spectrum of an Ame and an Ame-Zn complex;

FIG. 2 is a UV-vis spectrum of an Ame and an Ame-Zn complex;

FIG. 3 is a mass spectrum of an Ame;

FIG. 4a , FIG. 4b , FIG. 4c and FIG. 4d are mass spectrums of an Ame-Zncomplex;

FIG. 5 is a diagram showing results of inhibition effects of an Ame andan Ame-Zn complex on HegG2 cells;

FIG. 6 is a diagram showing results of inhibition effects of an Ame andan Ame-Zn complex on HeLa cells;

FIG. 7 is a diagram showing a result of an effect of fDNA on the UV-visspectrum of the Ame;

FIG. 8 is a diagram showing a result of an effect of fDNA on the UV-visspectrum of the Ame-Zn complex;

FIG. 9 is a diagram showing a result of an effect of an Ame on afluorescence emission spectrum of an fDNA-EB system;

FIG. 10 is a diagram showing a result of an effect of an Ame-Zn complexon a fluorescence emission spectrum of an fDNA-EB system;

FIG. 11 is a diagram showing a result of an effect of an Ame and anAme-Zn complex on a viscosity of an fDNA solution;

FIG. 12 is a diagram showing a result of an effect of an Ame on anauto-oxidation rate of pyrogallol;

FIG. 13 is a diagram showing a result of an effect of an Ame-Zn complexon an auto-oxidation rate of pyrogallol;

FIG. 14 is a diagram showing a result of an ABTS⁺. free radicalscavenging ability of an Ame; and

FIG. 15 is a diagram showing a result of an ABTS⁺. free radicalscavenging ability of an Ame-Zn complex.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention will be further described below in conjunctionwith specific embodiments.

Embodiment 1

53.8 mg (0.1 mmol) of Ame was accurately weighted and put in a roundbottom flask, then the Ame was dissolved with 5 mL of ethanol; 14.9 mg(0.05 mmol) of zinc nitrate hexahydrate was precisely weighted anddissolved with 5 mL of ethanol; the zinc nitrate solution was dropwiseadded to the Ame solution, ethanol-ammonia (V/V, 3:1) solution wasdropwise added to the reaction solution to adjust the pH to 6, and thereaction was performed at 30° C. for 4-5 h to obtain a precipitate; theprecipitate was filtered, and then successively washed with ethanol andwater, after that, recrystallized with the DMSO, and finallyfreeze-drying was performed to obtain the Ame-Zn complex.

Embodiment 2

53.8 mg of Ame was accurately weighted and put in a round bottom flask,then the Ame was dissolved with 5 mL of 90% ethanol; 14.9 mg of zincnitrate hexahydrate was precisely weighted and dissolved with 5 mL of90% ethanol; the zinc nitrate solution was dropwise added to the Amesolution, ethanol-sodium ethoxide solution was dropwise added to thereaction solution to adjust the pH to 5, and the reaction was performedat 30° C. for 4-5 h to obtain a precipitate; the precipitate wasfiltered, and then successively washed with ethanol and water, afterthat, recrystallized with the DMSO, and finally freeze-drying wasperformed to obtain the Ame-Zn complex.

Embodiment 3

53.8 mg of Ame was accurately weighted and put in a round bottom flask,then the Ame was dissolved with 5 mL of methanol; 14.9 mg of zincnitrate hexahydrate was precisely weighted and dissolved with 5 mL ofmethanol; the zinc nitrate solution was dropwise added to the Amesolution, methanol-sodium methoxide solution was dropwise added to thereaction solution to adjust the pH to 7, and the reaction was performedat 40° C. for 3-4 h to obtain a precipitate; the precipitate wasfiltered, and then successively washed with methanol and water, afterthat, recrystallized with the DMSO, and finally freeze-drying wasperformed to obtain the Ame-Zn complex.

Embodiment 4

53.8 mg of Ame was accurately weighted and put in a round bottom flask,then the Ame was dissolved with 5 mL of 85% methanol; 14.9 mg of zincnitrate hexahydrate was precisely weighted and dissolved with 5 mL of85% methanol; the zinc nitrate solution was dropwise added to the Amesolution, methanol-sodium methoxide solution was dropwise added to thereaction solution to adjust the pH to 7, and the reaction was performedat 50° C. for 2-3 h to obtain a precipitate; the precipitate wasfiltered, and then successively washed with methanol and water, afterthat, recrystallized with the DMSO, and finally freeze-drying wasperformed to obtain the Ame-Zn complex.

Embodiment 5

53.8 mg of Ame was accurately weighted and put in a round bottom flask,then the Ame was dissolved with 5 mL of ethanol; 6.8 mg of zinc chloridewas precisely weighted and dissolved with 5 mL of ethanol; the zincchloride solution was dropwise added to the Ame solution,ethanol-ammonia (V/V, 3:1) solution was dropwise added to the reactionsolution to adjust the pH to 6, and the reaction was performed at 30° C.for 4-5 h to obtain a precipitate; the precipitate was filtered, andthen successively washed with ethanol and water, after that,recrystallized with the DMSO, and finally freeze-drying was performed toobtain the Ame-Zn complex.

Embodiment 6

53.8 mg of Ame was accurately weighted and put in a round bottom flask,then the Ame was dissolved with 5 mL of 90% ethanol; 6.8 mg of zincchloride was precisely weighted and dissolved with 5 mL of 90% ethanol;the zinc chloride solution was dropwise added to the Ame solution,ethanol-sodium ethoxide solution was dropwise added to the reactionsolution to adjust the pH to 5, and the reaction was performed at 30° C.for 4-5 h to obtain a precipitate; the precipitate was filtered, andthen successively washed with ethanol and water, after that,recrystallized with the DMSO, and finally freeze-drying was performed toobtain the Ame-Zn complex.

Embodiment 7

53.8 mg of Ame was accurately weighted and put in a round bottom flask,then the Ame was dissolved with 5 mL of methanol; 6.8 mg of zincchloride was precisely weighted and dissolved with 5 mL of methanol; thezinc chloride solution was dropwise added to the Ame solution,methanol-sodium methoxide solution was dropwise added to the reactionsolution to adjust the pH to 7, and the reaction was performed at 40° C.for 3-4 h to obtain a precipitate; the precipitate was filtered, andthen successively washed with methanol and water, after that,recrystallized with the DMSO, and finally freeze-drying was performed toobtain the Ame-Zn complex.

Embodiment 8

53.8 mg of Ame was accurately weighted and put in a round bottom flask,then the Ame was dissolved with 5 mL of 85% methanol; 6.8 mg of zincchloride was precisely weighted and dissolved with 5 mL of 85% methanol;the zinc chloride solution was dropwise added to the Ame solution,methanol-sodium methoxide solution was dropwise added to the reactionsolution to adjust the pH to 7, and the reaction was performed at 50° C.for 2-3 h to obtain a precipitate; the precipitate was filtered, andthen successively washed with methanol and water, after that,recrystallized with the DMSO, and finally freeze-drying was performed toobtain the Ame-Zn complex.

Embodiment 9

The products prepared in Embodiments 1-8 were characterized. The IRspectra of the Ame and the Ame-Zn complex are shown in FIG. 1. As can beseen from the figure, the Ame has a broad absorption at 2800-3500 cm⁻¹,which is the stretching vibration peak of the associated hydroxyl group,because intramolecular hydrogen bonds are formed between 5-OH and 4-C═O,and between 5″-OH and 4″-C═0 in the Ame molecule. However, in the Ame-Zncomplex, the absorption is narrowed here, and the peak shape at about3400 cm⁻¹ becomes sharp, indicating that the intramolecular hydrogenbond is destroyed after the formation of the complex. The strong peak at1657 cm⁻¹ in the Ame is caused by the stretching vibration of thecarbonyl group, which is the characteristic absorption peak of thecarbonyl group. After the complex is formed, the absorption peak movestoward the low wave number and moves to 1631 cm⁻¹, indicating that thecarbonyl group participates in the coordination. The complex produces anabsorption peak between 614 cm⁻¹, which is caused by the stretchingvibration of Zn—O, effectively proving that oxygen atoms participate inthe coordination. Therefore, it can be speculated that the carbonylgroup and the hydroxyl group in the Ame participate in the coordination,and the most probable coordination sites are 5-OH, 4-C═O, 5″-OH and4″-C═O.

The UV-vis spectra of the Ame and the Ame-Zn complex are shown in FIG.2. The Ame has two characteristic absorption peaks at 337 nm (band I)and 270 nm (band II), which are the characteristic absorptions of theflavonoid compounds. Band I and band II correspond to the UV absorptionsof the cinnamyl system and the benzoyl system, respectively, which arecaused by the transition of π-π*. The Ame-Zn complex produces anabsorption platform between 375-450 nm, which is caused by the red shiftof the band I, indicating that the cinnamyl system participates in thecoordination. Although the position of the band II does not changesignificantly, the absorption intensity decreased relatively, and a newabsorption peak is also produced at 298 nm. These phenomena indicatethat the carbonyl group of the cinnamyl system and the benzoyl systemparticipates in the coordination. After the coordination, the conjugatedsystem increases, the energy required for the electronic transitiondecreases, and π-π* transition is more likely to occur, so the band I isred-shifted. However, 4-C═0 is more prone to trigger n-π* transition, soa new peak is generated at 298 nm. It can be speculated that the sitewhere Zn′ forms a complex with Ame is 5-OH, 4-C═O, 5″-OH, and 4″-C═O.

In the positive ion mode, the mass spectrometric analysis of the Ame andAme-Zn complex were carried out, and the ion structure corresponding tothe molecular ion peaks on the spectrum was obtained by simulationaccording to mass spectrometry, thus the structure of the Ame-Zn wasspeculated.

FIG. 3 is a mass spectrum of the Ame in the positive ion mode with thequasi-molecule ion peak m/z 539.0920 belonging to [Ame+H]⁺. FIG. 4a is amass spectrum of the Ame-Zn complex, which shows that the quasi-moleculeion peak of the Ame-Zn complex is an ion peak m/z 757.0417 with apositive charge. From its isotope mass spectrum (FIG. 4b ), it can beseen that the isotope peaks of the quasi-molecule ion peaks are mainlym/z 758.0444, m/z 759.0385, m/z 760.0402, m/z 761.0364, m/z 762.0385,and m/z 763.0412, and the molecular weights of adjacent ion peaks differby 1.0027, 0.9941, 1.0017, 0.9962, 1.0021, and 1.0027, respectively,confirming that the ion carries a positive charge. It can be seen fromthe IR spectrum and the UV-vis spectrum that the coordination siteswhere the Ame forms complex with Zn²⁺ are 5-OH, 4-C═O, 5″-OH and 4″-C═O.Since the solvent for recrystallization and dissolution of the complexis DMSO, DMSO contains an oxygen atom and a sulfur atom, which has astrong coordination ability and is difficult to ionize, the complex maycontain DMSO, thus speculating that the quasi-molecule ion peak m/z757.0417 belongs to [Zn(Ame-H)(DMSO)₂]⁺. The elemental composition isC₃₄H₂₉O₁₂ZnS₂, which is consistent with the possible elementalcomposition of the quasi-molecule ion peak m/z 757.0417 (FIG. 4c ).Moreover, [Zn(Ame-H)(DMSO)₂]⁺ was simulated by mass spectrometrysimulation software to obtain the simulated mass spectrum as shown inFIG. 4d . It can be seen from the figure that the isotope ion peaks of[Zn(Ame-H)(DMSO)₂]⁺ are m/z 757.0386, m/z 758.0420, m/z 759.0356, m/z760.0389, m/z 761.0343, m/z 762.0377, m/z 763.0301, respectively, whichare highly matched to the isotope mass spectrum peak of thequasi-molecule ion peak m/z 757.0417. Therefore, it can be confirmedthat the ion corresponding to the molecular ion peak m/z 757.0417 is[Zn(Ame-H)(DMSO)₂]⁺. In summary, Ame and Zn²⁺ form a complex in a ratioof 1:1. The coordination sites where Ame forms complex with Zn²⁺ areconfirmed to be 5-OH, 4-C═O, 5″-OH and 4″-C═O by IR spectrum and UV-visspectrum. Since the activity of 5″-OH is higher than that of 5-OH, thecoordination sites where Ame forms complex with Zn²⁺ are the most likelyto be 5″-OH and 4″-C═O. Therefore, the most likely structural formula of[Zn(Ame-H)(DMSO)₂]⁺ is as follows:

In addition, since the metal salt in the experiment is a nitrate or ahydrochloride, the complex contains nitrate or chloride ions. Therefore,the structural formula of the Ame-Zn complex is as follows:

X is NO₃ ⁻ or Cl⁻.

Embodiment 10

The antitumor activity of Ame and Ame-Zn complex were studied by MTTmethod with the following process:

(1) the HepG2 and HeLa cell suspensions were inoculated into 96-wellculture plates, 100 μL was added for each well, (1×10⁵/mL), and thencultured in a 5% CO₂ incubator at 37° C. for 24 h;

(2) after culturing for 24 h, the supernatant was discarded, 100 μL ofpre-diluted sample was added, 10 replicate wells were set for eachconcentration, and then incubated in the 5% CO₂ incubator for 24 h;meanwhile, control wells (DMSO, cell suspension, MTT), and blank wells(medium, DMSO, MTT) were set;

(3) After culturing for 36 h, the supernatant was discarded, 100 μL ofDMEM medium containing MTT (5 mg/mL) were added, and then continuouslycultured for 4 h;

(4) After 4 h, the supernatant was carefully scavenged, 200 μL of DMSOwas added to each well, adequate shaking was performed for 15 min in aconstant temperature oscillator, the absorbance was measured at 595 nmby a microplate reader, the inhibition rate of the sample to HepG2 andHeLa cells was calculated by OD, and the half-inhibitory concentrationIC₅₀ value was calculated by using the modified Karber formula.

$\begin{matrix}{{IR} = {1 - \frac{{OD}_{1}}{{OD}_{0}}}} & \left( {{Formula}\mspace{14mu} 1} \right) \\{{lgIC}_{50} = {{Xm} - {l\left( {P - {\left( {3 - {Pm} - {Pn}} \right)/4}} \right)}}} & \left( {{Formula}\mspace{14mu} 2} \right)\end{matrix}$

Where, IR is the inhibition rate, OD₀ is the absorbance of the controlgroup, OD₁ is the absorbance of the sample group, Xm is 1 g (the maximumdose), I is 1 g (the maximum dose/adjacent dose), P is the sum ofpositive reaction rates, Pm is the maximum positive reaction rate, andPn is the minimum positive reaction rate.

The results are shown in FIGS. 5-6. It is found that Ame-Zn complex caneffectively inhibit the growth of the hepatoma cells (HepG2) and thecervical cancer cells (HeLa), IC₅₀ values thereof are 5.359 and 5.119μmol·L⁻¹, respectively, which is smaller than the IC₅₀ values of the Ame(The IC₅₀ values of Ame are 13.633 and 8.040 μmol·L⁻¹, respectively),indicating that the Ame-Zn complex has better antitumor activity thanthe Ame. UV-vis spectroscopy, fluorescence spectroscopy and viscositymethod are used to study the interaction between the Ame/Ame-Zn complexand herring sperm DNA (fDNA) to further reveal the mechanism ofantitumor activity, the obtained spectra are shown in FIGS. 7-11. Theresults show that the interaction between the Ame/Ame-Zn complex and thefDNA is in the intercalation manner, and the interaction between thecomplex and the fDNA is stronger than that between the Ame and the fDNA.Thereby, it can be speculated that the mechanism of antitumor activityof the Ame and its complex may be that Ame or the complex thereof entersthe interior of the cell and intercalates with the DNA strand to causeapoptosis. Since the interaction of the complex with DNA is strongerthan of the Ame, the antitumor activity thereof is also stronger than ofthe Ame.

Embodiment 11

The free radical scavenging effects of the Ame and the Ame-Zn complexwere studied by pyrogallol auto-oxidation method and ABTS method withthe following steps:

(1) Pyrogallol Auto-Oxidation Method

Determination of auto-oxidation rate V₀ of pyrogallol: 2 mL Tris-HClbuffer (pH=8.20) was added to a 10 mL sample tube at 25° C., 100 μL DMSOwas added as a control, after adding 0.8 mL distilled water, 0.2 mLpyrogallol solution having a concentration of 2 mmol·L⁻¹ was added, themixture was poured into a cuvette after mixing uniformly, the absorbanceat 322 nm was measured with pure water as a blank, the A value wasrecorded every 10 s for a total of 4 min, linear regression wasperformed with t as the abscissa, and A as the ordinate, straight lineslope thereof is V₀, and the measurement was performed for three timesto obtain an average value.

Determination of auto-oxidation rate Vi of pyrogallol after addingsample: 2 mL Tris-HCl buffer (pH=8.20) was added to a 10 mL sample tubeat 25° C., 100 μL samples of various concentrations that were dissolvedin DMSO was added, after adding 0.8 mL distilled water, 0.2 mLpyrogallol solution having a concentration of 2 mmol·L⁻¹ was added, themixture was poured into a cuvette after mixing uniformly, the absorbanceat 322 nm was measured with double distilled water as a blank, the Avalue was recorded every 10 s for a total of 4 min, linear regressionwas performed with t as the abscissa, and A as the ordinate, straightline slope thereof is Vi, and the measurement was performed for threetimes to obtain an average value. The free radical scavenging rate wascalculated according to Formula 3.SR(%)=(1−v ₁ /v ₀)×100%  (Formula 3)

(2) ABTS Method

Determination of ABTS⁻. free radical ion scavenging capacity of theblank sample: at 25° C., 2.9 mL ABTS⁺. free radical ion working solutionwas added into a 10 mL sample tube, 100 μL DMSO was added, afterreacting for 5 min, the UV-vis spectrum was measured, and the absorptionintensity A₀ at 730 nm was recorded.

Determination of ABTS⁺. free radical ion scavenging capacity of thesamples: at 25° C., 2.9 mL ABTS⁺. free radical ion working solution wasadded into a 10 mL sample tube, 100 μL samples of differentconcentrations that were dissolved in DMSO were added, after reactingfor 5 min, the UV-vis spectrum was measured, and the absorptionintensity A₁ at 730 nm was recorded, and the ABTS⁺. free radical ionscavenging rate was calculated according to Formula 4.SR(%)=(1−A ₁ /A ₀)×100%  (Formula 4)As shown in FIGS. 12-15, the results of pyrogallol auto-oxidation methodshow that the IC₅₀ of the Ame and the Ame-Zn complex to scavenge O₂ ⁻.free radical are 23.273 μmol·L⁻¹ and 7.842 μmol·L⁻¹, and it can be foundthat the O₂ ⁻. free radical scavenging ability of the Ame-Zn complex issignificantly stronger than that of the Ame.

The ABTS⁺. free radical scavenging capacities of the Ame and the Ame-Zncomplex are concentration dependent. Within a certain range, thescavenging rate is linear with the concentration. The curve of thescavenging rate and the concentration (c) is plotted in the presentinvention to obtain a corresponding linear equation, and the maximumhalf-inhibitory concentration (IC₅₀ value) is calculated. The IC₅₀values of the Ame and the Ame-Zn complex for scavenging ABTS⁺. freeradicals are 20.703 and 7.077 μmol·L⁻¹, respectively. It can be seenthat the ABTS⁺. free radical scavenging ability of the Ame-Zn complex issignificantly stronger than that of the Ame.

What is claimed is:
 1. A biflavone-zinc complex, comprising the following structural formula:

wherein, X is NO₃ ⁻ or Cl⁻.
 2. A preparation method of the biflavone-zinc complex of claim 1, wherein a zinc salt is dissolved in an alcohol and added into a biflavone dissolved in an alcohol to obtain a mixed solution, pH of the mixed solution is controlled to 5-7, under heating and stirring the mixed solution, a reacting of the mixed solution is performed for 2-5 h to form a precipitate; the precipitate is filtered, and then washed with alcohol and water, after that recrystallized using dimethyl sulfoxide as a solvent, and finally drying is performed to the precipitate to obtain the biflavone-zinc complex.
 3. The preparation method of the biflavone-zinc complex of claim 2, wherein the biflavone is an amentoflavone.
 4. The preparation method of the biflavone-zinc complex of claim 2, wherein the zinc salt is zinc nitrate or zinc chloride.
 5. The preparation method of the biflavone-zinc complex of claim 2, wherein the pH is adjusted with an alkali alcohol solution, and an alkali used in the alkali alcohol solution is selected from the group consisting of sodium hydroxide, potassium hydroxide, aqueous ammonia, sodium ethoxide, and sodium methoxide.
 6. The preparation method of the biflavone-zinc complex of claim 2, wherein a reaction temperature of the reacting is 30° C.-50° C., and a reaction time of the reacting is 2-5 h.
 7. The preparation method of the biflavone-zinc complex of claim 2, wherein a molar ratio of the biflavone to zinc ion in the solution is 2-2.5:1.
 8. The preparation method of the biflavone-zinc complex of claim 2, wherein the solvent used in the recrystallizing is dimethyl sulfoxide, and a method of the drying is freeze-drying or low temperature vacuum-drying. 